Mu-opioid Receptor Gene Methylation Correlates with Increased Opioid Use and Cancer Pain
Methods: Seventy-three patients with cancer were enrolled with the following inclusion criteria: 1) biopsy-proven cancer, 2) able to read, write and understand English, 3) Karnofsky Performance Status (KPS) score ≥60, 4) scheduled for primary or adjuvant radiotherapy (RT), and 5) prescribed opioids for cancer pain management. DNA was extracted from blood collected prior to RT. OPRM1 methylation was quantified using the EpiTYPER assay.
An orthotopic mouse cancer model was created by inoculating HSC-3, a tongue squamous cell carcinoma cell line (SCC), into the right hind paw of BALB/c mice. Adenoviral transduction was used to re-express either OPRM1 or GFP in the paw tumor. Mechanical allodynia and thermal hyperalgesia were quantified at baseline and on post-inoculation day (PID) 14, and after local naloxone administration. Immunohistochemistry and RT-PCR were performed to detect mu-opioid receptor expression in paw tumors and associated dorsal root ganglia (DRG).
Beta-endorphin was measured in vitro in HSC-3 cells after adenoviral transduction of either OPRM1 or GFP, and after naloxone treatment.
Results: Patients were divided into either: 1) no-use, 2) low-dose or 3) high-dose opioid users. The low-dose group used between100mg and 200mg morphine equivalent total for days 1 and 2 after enrollment. High-dose group patients were more likely to have OPRM1 promoter hypermethylation than the low-dose group. While there is no association between level of pain relief and amount of opioid used, patients with inadequate pain relief were more likely to have OPRM1 hypermethylation than patients with near-total pain relief.
Mice with paw tumors transduced with Ad-OPRM1 had less mechanical allodynia and thermal hyperalgesia than control mice transduced with Ad-GFP. The mechanical and thermal antinociceptive effects were reversible by naloxone. Mice in the Ad-GFP group had no response to naloxone, suggesting that the mu-opioid receptor and endogenous opioids are necessary for the antinociceptive effect.
While SCC paw tumors suppressed mu-opioid receptor expression in the associated DRG, re-expression of the receptor in the paw tumors led to a reversal of suppression so that the associated DRGs demonstrated normal mu-opioid receptor expression. Re-expression of mu-opioid receptor in SCC cells in vitro resulted in an increase in secretion of the endogenous opioid beta-endorphin, which was reversible by naloxone.
Conclusions: In this study we show for the first time that mu-opioid receptor expression on cancer cells produces analgesia. The analgesic effect is likely due to two mechanisms: 1) increased secretion of beta-endorphin into the cancer microenvironment, and 2) restoration of mu-opioid receptors on the sensory neurons.